FAQ's

Different promoters work better with certain cell types. CMV, though a strong promoter, may not be optimal for all cell types. For example, EF-1α is a better promoter for gene expression in stem cells.

Complexity of the protein being expressed can affect efficiency. Small proteins with limited tertiary structure (like GFP) are generally more easily expressed by cells. Also with a large complex construct, you may need to alter time of analysis post-transfection.

DNA, even if stored appropriately, can still degrade over time. Verify that you are using full length DNA.